This project involves a multi-disciplinary research effort directed at structure/function/inhibition studies of the enzyme thymidylate synthase (TS). This enzyme has gained increased interest over the past five years because of several major accomplishments/findings: (i) The X-ray crystal structures of TS from several different sources and in different bound forms have been solved. Now, structures of mutants can be readily solved by molecular replacement. (ii) The human TS has been expressed. (iii) The Lactobacillus casei TS gene has been chemically synthesized, serving as an ideal mutagenesis/expression vector. (iv) Conditions have been found to unfold/refold TS. Our studies of TS can be subdivided into several categories: (1) We are carrying out structure-function studies of TS using a mutational approach. Here, we mutate a chosen amino acid to all 19 other residues using "mixture-cassette mutagenesis" of the synthetic gene; a segment of the synthetic gene is replaced by mixtures of oligonucleotides containing all codons at the target site. The mutants are identified, individually purified and characterized. X-ray and other biophysical studies are undertaken for the interesting mutants. A similar approach is taken to prepare combinatorial (multiple) mutations, including combinatorial libraries. (2) In other studies, we are attempting the rational design of peptide and other inhibitors of TS with unique modes of action. one approach will utilize computational methods for the design and development of novel inhibitors. (3) We are performing folding studies of TS mutants to try to understand molecular features of subunit dimerization. (4) We are attempting to determine the PKa Of the catalytic thiol by 13C NMR. Mass Spectrometry is used for analysis and identification of compounds which are potential inhibitors of TS or products of the reaction of TS with unusual inhibitors. It is also used in the analysis of mutant enzymes and confirmation of predicted molecular weights. In the case of the human enzyme, a cleaved enzyme is formed under some conditions. We are using Mass spec to determine the exact molecular weight in order to identify the cleavage site.